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1.
Biotechnol Bioeng ; 81(7): 800-8, 2003 Mar 30.
Article in English | MEDLINE | ID: mdl-12557313

ABSTRACT

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.


Subject(s)
Biotin/analogs & derivatives , Cell Aggregation/drug effects , Cell Membrane/metabolism , Myoblasts/drug effects , Myoblasts/physiology , Periodic Acid/pharmacology , Aldehydes/metabolism , Animals , Biotin/metabolism , Cell Aggregation/physiology , Cell Count , Cell Membrane/drug effects , Cell Survival/drug effects , Cells, Cultured , Feasibility Studies , Flow Cytometry , Hexosamines/pharmacology , Ketones/metabolism , Membrane Proteins/metabolism , Myoblasts/cytology , Myoblasts/ultrastructure , Oxidation-Reduction , Rats , Tissue Engineering/methods
2.
Arch Biochem Biophys ; 317(2): 439-48, 1995 Mar 10.
Article in English | MEDLINE | ID: mdl-7893161

ABSTRACT

We have examined and characterized the binding of transformed guinea pig hepatic Ah receptor to its specific DNA recognition site, the dioxin-responsive element (DRE), using gel retardation analysis. Saturation binding analysis of transformed TCDD:AhR complexes were indicative of a single high-affinity binding site (Kd = 2.5 +/- 0.8 nM); however, DNA-binding analysis revealed the presence of two distinct TCDD-inducible protein-DRE complexes. Sucrose gradient centrifugation and subsequent gel retardation analysis of the fractions demonstrated a similarity in the distribution of [3H]TCDD-specific binding and TCDD-inducible protein-DNA complex formation, supporting the presence of the AhR in both complexes. In addition, the formation of both DNA-binding complexes exhibited the same nucleotide specificity previously determined for the AhR complex. Since labeling studies using a radio-iodinated photoaffinity dioxin agonist demonstrated that guinea pig cytosol contains a single ligand binding subunit of 105 kDa, the difference in migration of the complexes is due to other proteins associated with each complex. Overall, our results demonstrate the presence of two distinct high affinity DNA-binding forms of transformed guinea pig AhR complex which exhibit similar DNA-binding affinity and nucleotide specificity.


Subject(s)
DNA/metabolism , Liver/chemistry , Receptors, Aryl Hydrocarbon/metabolism , Animals , Base Sequence , Binding Sites , Binding, Competitive , Centrifugation, Density Gradient , Chromatography, Gel , Cross-Linking Reagents , Cytosol/chemistry , DNA/chemistry , Electrophoresis, Polyacrylamide Gel , Guinea Pigs , Male , Molecular Sequence Data , Polychlorinated Dibenzodioxins/metabolism , Polychlorinated Dibenzodioxins/pharmacology , Rats , Receptors, Aryl Hydrocarbon/chemistry , Receptors, Aryl Hydrocarbon/genetics , Ultraviolet Rays
3.
Eur J Pharmacol ; 228(2-3): 85-94, 1992 Sep 01.
Article in English | MEDLINE | ID: mdl-1332880

ABSTRACT

The Ah receptor (AhR) mediates many, if not all, of the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons. Although wide variations in species sensitivity to these compounds have been observed, numerous biochemical and physiochemical characteristics of the AhR appear similar among species. We have examined the ability of cytosolic AhR, from a variety of species (rat, rabbit, guinea pig, hamster, mouse, cow, sheep, fish, chicken and human), to transform and bind to its cognate DNA recognition sequence, the dioxin responsive enhancer (DRE), to evaluate the importance of these events in species variations in TCDD responsiveness. Gel retardation analysis using a murine DRE oligonucleotide has revealed that cytosolic AhR from a wide variety of species can transform in vitro and bind to the DRE and demonstrates that all of the factors necessary for AhR transformation and DNA binding are present in cytosol. In addition, DNA-binding analysis using a series of mutant DRE oligonucleotides has indicated no apparent species- or ligand-dependent, nucleotide-specific difference in AhR binding to the DRE. These studies support a highly conserved nature of the DRE and AhR (at least in DNA binding) and imply that a sequence closely related to the murine consensus DRE sequence is responsible for conferring AhR-dependent, TCDD responsiveness in each of these species.


Subject(s)
Polychlorinated Dibenzodioxins/metabolism , Receptors, Drug/metabolism , Animals , Base Sequence , Binding, Competitive , Cattle , Cells, Cultured , Cricetinae , Cytosol/drug effects , Cytosol/metabolism , Guinea Pigs , Humans , Male , Mesocricetus , Mice , Mice, Inbred CBA , Molecular Sequence Data , Polychlorinated Dibenzodioxins/pharmacology , Rabbits , Rats , Rats, Sprague-Dawley , Receptors, Aryl Hydrocarbon , Species Specificity , Swine , Transcription Factors , Trout
4.
Biochim Biophys Acta ; 993(1): 1-6, 1989 Oct 13.
Article in English | MEDLINE | ID: mdl-2508757

ABSTRACT

Administration of a single oral dose (10 micrograms/kg) of tetrachlorodibenzo-p-dioxin (TCDD) caused a 33% decrease in retinyl esters in the livers of male rats, but a 13-fold increase in retinyl esters in the kidney and a 3-fold increase in serum retinol. Liver and kidney microsomal uridine diphosphoglucuronosyltransferase (UDPGT) activity toward all-trans-retinoic acid was increased 3.7- and 2.6-fold, respectively, ten days following exposure to TCDD. Verification of the in vitro formation of [3H]retinoyl beta-glucuronide (RG) was by cochromatography with authenic RG on reversed phase high pressure liquid chromatography (HPLC), identification of retinoic acid as the hydrolysis product after beta-glucuronidase treatment, and the characterization of the all-trans-retinoyl glucuronide by negative fragment mass spectroscopy, fast atom bobardment. We conclude that increased retinoic acid glucuronidation may be a contributing factor to the hepatic depletion of vitamin A and the increased excretion of vitamin A metabolites following TCDD exposure.


Subject(s)
Dioxins/pharmacology , Glucuronosyltransferase/metabolism , Kidney/metabolism , Liver/metabolism , Microsomes, Liver/metabolism , Polychlorinated Dibenzodioxins/pharmacology , Tretinoin/analogs & derivatives , Tretinoin/metabolism , Vitamin A/metabolism , Animals , Chromatography, High Pressure Liquid , Kidney/drug effects , Kinetics , Liver/drug effects , Male , Mass Spectrometry , Rats , Rats, Inbred Strains , Reference Values , Vitamin A/isolation & purification , Vitamin A Deficiency/metabolism
5.
Z Ernahrungswiss ; 28(2): 93-102, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2763594

ABSTRACT

Adequate stores and adequate tissue levels of vitamin A are maintained by a balance of tissue demands and dietary intake of the vitamin and are modified by many factors, including xenobiotics. It is well established that exposure to polyhalogenated aromatic hydrocarbons (PHAH) decreases hepatic content of vitamin A. Recent findings indicate that hepatic depletion of vitamin A is accompanied by an increase in serum and renal vitamin A content and enhanced excretion of vitamin A metabolites in urine and feces. Examination of tissue retinoid profiles reveals that PHAH exposure causes the generation of increased amounts of polar retinoids. It is very likely that PHAH affect enzymes crucial for regulation of vitamin A storage as well as enhance activities of specific enzymes in vitamin A metabolic pathway.


Subject(s)
Hydrocarbons, Halogenated/toxicity , Vitamin A/pharmacokinetics , Animals , Biotransformation/drug effects , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Rats , Retinoids/pharmacokinetics , Tissue Distribution/drug effects
6.
Biochim Biophys Acta ; 990(3): 306-14, 1989 Mar 24.
Article in English | MEDLINE | ID: mdl-2923910

ABSTRACT

Vitamin A status and turnover were examined in rats that had been exposed to chronic dietary treatment of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 1 mg/kg diet. HCB caused hepatic depletion and renal accumulation of vitamin A, and a 1.7-fold increase in the serum retinol concentration. Intravenously administered [3H]retinol bound to retinol binding protein-transthyretin complex (RBP-TTR complex) was used to study the dynamics of circulatory retinol in these rats. In HCB-treated rats, the plasma turnover rate of retinol was increased compared to vitamin A-adequate untreated controls. HCB caused a 50% reduction of total radioactivity in liver, and, except for 0.5 h after the [3H]retinol-RBP-TTR dose, the specific activity of the hepatic retinyl ester pool was greater compared to control rats. The kidneys of HCB-treated rats accumulated radioactivity in the retinyl ester fraction. HCB also caused a 50% reduction in adrenal radioactivity compared with control rats. Urinary and fecal excretion of radioactivity was 3-fold higher in HCB-treated rats as compared to controls. Our findings demonstrate that chronic HCB feeding results in expansion of plasma vitamin A mass, in changes of liver and kidney retinol and retinyl ester pool dynamics and in an increased metabolism of vitamin A.


Subject(s)
Chlorobenzenes/toxicity , Hexachlorobenzene/toxicity , Homeostasis/drug effects , Vitamin A/pharmacokinetics , Animals , Feces/analysis , Female , Half-Life , Kidney/drug effects , Kidney/metabolism , Liver/drug effects , Liver/metabolism , Metabolic Clearance Rate/drug effects , Rats , Rats, Inbred Strains , Retinoids/blood , Tritium , Vitamin A/blood , Vitamin A/urine
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